Rapid and accurate detection and determination of creatinine, which is found in blood or urine, are very important for diagnosis of diseases such as uremia, chronic nephritis, acute nephritis, gigantism, and tonic muscular dystrophy.
A creatinine amide hydrolase (EC 3.5.2.10) has been used as an enzyme for determination of creatinine in body fluids, which is an indicator in clinical diagnosis of muscular and renal diseases, together with other enzymes such as creatine amidinohydrolase, sarcosine oxidase and peroxidase. A creatinine amide hydrolase is an enzyme which acts on creatinine to catalyze a reversible reaction of the formation of creatine from creatinine in the presence of water.
Such a creatinine amide hydrolase is known to be produced by microorganisms of the genera Pseudomonas (non-Patent Document 1) and Alcaligenes (non-Patent Document 2). In addition, as other producers of a creatinine amide hydrolase, only microorganisms of the genera Flavobacterium, Corynebacterium, Micrococcus (Patent Document 1), Penicillium (Patent Document 2), etc. are known. Among them, a gene encoding a creatinine amide hydrolase produced by Pseudomonas putida PS-7 was already isolated, and the amino acid sequence thereof has been published (Patent Document 3).
non-Patent Document 1: Journal of Biochemistry, Vol. 86, 1109-1117 (1979)
non-Patent Document 2: Chemical and Pharmaceutical Bulletin, Vol. 34, No. 1, 269-274 (1986)
Patent Document 1: JP 51-115989 A
Patent Document 2: JP 47-43281 A
Patent Document 3: Japanese Patent 2527035
However, the creatinine amide hydrolase produced from various known microorganisms had a large Km value to creatinine as an enzyme for a clinical-test and thus, a greater amount of the enzyme should be added to a reagent composition. For example, an enzyme derived from Alcaligenes faecalis TE3581 (Patent Document 4) is reported to have a Km value to creatinine of about 42 mM. Another enzyme derived from Arthrobacter sp. TE1826 has a larger Km value to creatinine of about 66 mM (Patent Document 5).
Patent Document 4: JP 9-154574 A
Patent Document 5: JP 10-215874 A
For quantitative determination of creatinine, known is a method of quantitatively determining creatinine in a sample by treating the creatinine in the sample with creatinine amide hydrolase, creatine amidinohydrolase and sarcosine oxidase and determining the resulting hydrogen peroxide by a hydrogen peroxide-determining means.
For carrying out such a method, a universally applicable automatic analyzer is often used, wherein a concentration of an objective substance is determined by dividing reagents into two or more portions, adding the portions one by one in a predetermined addition order to a reaction cell to react the portions for about several minutes to about 20 minute as the entire steps, monitoring an increase and a decrease in absorbance over time during the period, and analyzing and calculating the results. There are various known reagents that can be used in these automatic analyzers.
Non-patent Document 3: Medical Technology, Vol. 10, No. 7, 575-579 (1982)
However, in conventional methods, since it takes a longer period of time until the reaction reaches the end point, the number of samples to be analyzed is limited. On the other hand, for completion of the reaction in a short period of time, an amount of an enzyme to be added should be raised, which has caused an economical problem.